Psychostimulants, including cocaine as well as novel cocaine analogs developed by this Center, act by blocking transporter-mediated reuptake of biogenic amines (dopamine, 5-HT and norepinephrine). This project utilizes novel tropane and methylphenidate analogs, along with specific animals models developed by other Center projects, to study transporter binding sites, and to examine the consequences of chronic drug treatment on G-protein-coupled receptors. The first aim of this project utilizes animal models developed by the Center to examine how chronic treatment with psychostimulants (both cocaine and novel cocaine analogs) affect coupling of biogenic amine receptors to G-proteins in brain. These studies will utilize [35S]GTPgammaS autoradiography, which provides a neuroanatomical localization of the activation of G-protein receptors. Because this technique allows for an number of different receptors to be assayed simultaneously in brain sections from the same animal, it is an ideal method to efficiently analyze brain tissue from Center-derived animals. This project will follow up on previous studies showing that chronic opioid treatment produced selective attenuation of mu opioid-activated G-proteins in specific brainstem nuclei. Three different drug treatment paradigms will be employed: a chronic treatment with the highly potent tropane analog WF-23, chronic self-administration in a binge model of cocaine developed by Dr. Roberts in subproject 0011, and a speedball (heroin/cocaine combination) self-administration model developed by Drs. Smith and Martin in subproject 0005. The second aim will characterize the properties of novel irreversible cocaine analogs (both tropanes and methylphenidates) as probes of dopamine transporter structure and function. Since these compounds will be used in behavioral studies by other Center projects, our laboratory will first characterize the basic irreversible binding properties of these analogs both in vitro and in vivo, using transporter radioligand binding in membranes and autoradiography. The most potent analogs will be labeled with [1251] and differences in binding between tropanes and methylphenidates will be examined by SDS-PAGE and purification of [1251]-labeled tryptic peptide fragments isolated from cells transfected with dopamine transporters.